Crystallization and purification of macrolides

ABSTRACT

Provided is a method for crystallization and purification of a macrolide such as tacrolimus, sirolimus, pimecrolimus, or everolimus that includes the step of providing a combination of a macrolide, and a polar solvent, dopolar aprotic solvent, or hydrocarbon solvent at pH of 7 or above.

RELATED APPLICATIONS

[0001] The present application claims the benefit of the filing date ofthe following U.S. Provisional Patent Applications: U.S. provisionalapplication Serial No. 60/512,887, filed Oct, 20, 2003, U.S. provisionalapplication Serial No. 60/461,707, filed Apr. 9, 2003, and U.S.provisional application Serial No. 60/459,591, filed March 31, 2003, thecontents of all of which are incorporated herein.

FIELD OF THE INVENTION

[0002] The present invention relates to the crystallization andpurification of macrolides, especially tacrolimus, sirolimus(rapamycin), pimecrolimus, and everolimus.

SUMMARY OF THE INVENTION

[0003] The present invention relates to a method for crystallization andpurification of macrolides, especially tacrolimus, sirolimus,pimecrolimus, and everolimus, including the steps of: providing acombination of a macrolide starting material; a polar solvent,especially a polar solvent that is an alkyl ester of an alkanoic acid,an alcohol, an ether, an aliphatic ketone, an aliphatic nitrile, or adipolar aprotic solvent; a hydrocarbon solvent, especially an acyclic orcyclic aliphatic hydrocarbon or an aromatic hydrocarbon (e.g. toluene);and water; at a pH of about 7 or above, especially about 8 or above;maintaining the combination at a temperature of between about −15° C. toabout 50° C., preferably between about −5° C. to about 40° C. , mostpreferably between about −2° C. to about 35° C. for at least about 1hour, preferably between about 48 to about 100 hours; and isolatingcrystalline macrolide.

[0004] In another aspect, the present invention relates to a method forcrystallization and purification of a macrolide, especially tacrolimus,sirolimus, pimecrolimus, or everolimus including the steps of: providinga concentrate residue from whole-broth extraction ofmacrolide-containing biomatter in a polar solvent, especially a polarsolvent that is an alkyl ester of an alkanoic acid, an alcohol, anether, an aliphatic ketone, an aliphatic nitrile, or a dipolar aproticsolvent; combining the solution, in any order, with water and ahydrocarbon solvent, especially an acyclic or cyclic aliphatichydrocarbon or an aromatic hydrocarbon (e.g. toluene), wherein the pH isabout 7 or above, especially about 8 or above; maintaining thecombination at a crystallization temperature for a crystallization time;and isolating crystalline macrolide.

[0005] In a further aspect, the present invention relates to a method ofcrystallizing and purifying a macrolide, especially tacrolimus,sirolimus, pimecrolimus, or everolimus including the steps of:combining, in any order, an oil that is a concentrate obtained byconcentrating a solution obtained by extracting macrolide-containingbiomatter with a hydrophobic extraction solvent, e.g. butyl acetate;with a polar solvent, especially a polar solvent that is an alkyl esterof an alkanoic acid, an alcohol, an ether, an aliphatic ketone, analiphatic nitrile, or a dipolar aprotic solvent; a hydrocarbon solvent,especially an acyclic or cyclic aliphatic hydrocarbon or an aromatichydrocarbon (e.g. toluene); and water; wherein the pH is about 7 orabove, especially 8 or above; maintaining the combination at a firstcrystallization temperature for a first crystallization time; andisolating crystalline macrolide.

[0006] In any of the forgoing aspects, the combination can be, but neednot be, maintained at a second crystallization temperature for a secondcrystallization time.

DETAILED DESCRIPTION OF THE INVENTION

[0007] As used herein in connection with a measured quantity, “about”refers to that variation in the measured quantity as would be expectedby the skilled artisan performing or interpreting the measurement andexercising a level of care commensurate with the objective of themeasurement and the precision of the measuring equipment being used.

[0008] As used herein, ambient temperature refers to a temperature ofabout 18° C. to about 25° C.

[0009] As used herein, “RN” refers to the registry number assigned to achemical compound by the Chemical Abstracts Service, Columbus Ohio,USA).

[0010] The method of the present invention is applied to thecrystallization and purification of macrolides from macrolide-containingstarting material. The macrolides are multi-membered lactone ringshaving one or more deoxy sugars as substituents. Erythromycin,azithromycin, and clarithromycin are macrolides that have bacteriostaticand/or bactericidal activity. The macrolides tacrolimus (FK 506) andsirolimus (rapamycin) are preferred macrolides for use in the practiceof the present invention. The macrolides pimecrolimus (the 33-epichloroderivative of ascomycin; RN=137071-32-0) and everolimus(40-O-(2-hydroxyethyl)-rapamycin; RN=159351-69-6) are also preferredmacrolides for use in the practice of the present invention.

[0011] The macrolides are typically obtained by fermentation, althoughsynthetic routes to some are known. The macrolide starting material foruse in the practice of the present invention can be from any source.Concentrate residue from concentrating the extract of the entirefermentation broth (“whole broth method”) from macrolide-containingbiomatter can be used as the macrolide starting material for the presentmethod. Use of hydrophobic extraction solvent in the extraction toobtain solution to be concentrated results in an efficient extractionyield, leaving behind most water-soluble impurities, with removal ofmycelium in one step. Concentration under reduced pressure at T>25° C.and reduced pressure results in a high evaporation rate of solventwithout precipitation or decomposition of macrolide and provides amacrolide starting material for use in the practice of the presentinvention. Concentrate residue for use as macrolide starting material inthe practice of the present invention can be obtained as described inU.S. patent application Ser. No. 10/366,266, published as U.S.2003/01666924 A1 and incorporated herein in its entirety by reference.

[0012] Oily residue from macrolide-producing procedsses can also be usedas starting macrolide starting material.

[0013] Preferred macrolide-containing biomatter that can be a source ofmacrolide starting material for the practice of the present inventionincludes tacrolimus-containing biomatter, particularly fermentationbroth obtainable by fermentation using a tacrolimus-producingmicroorganism, for example, Streptomyces tsukubaensis, new and mutatedstrains thereof, Streptomyces hygroscopicus, and Streptomyces lividans,as described in U.S. Pat. Nos. 4,894,366, 5,116,756, 5,624,842,5,496,727, and 5,622,866, all of which are incorporated herein byreference. Sirolimus-containing (rapamycin-containing) biomatter is alsoa preferred macrolide-containing biomatter. Sirolimus (rapamycin) can beproduced by fermentation of Streptomyces hygroscopicus, NRRL 5491, asdescribed in U.S. Pat. No. 3,993,749, incorporated herein by reference.Pimecrolimus-containing biomatter and everolimus-containing biomatterare also examples of preferred macrolide-containing biomatter for use inthe practice of the method of the present invention.Ascomycin-conyaining biomatter is also a preferred macrolide-containingbiomatter for use in the practice of the present invention

[0014] The method of the present invention employs, among other things,polar solvents, hydrocarbon solvents, and bases (alkali).

[0015] Polar solvents are organic compounds, normally liquid at ambienttemperature, that dissolve a macrolide, especially tacrolimus,sirolimus, pimecrolimus, or everolimus. Polar solvents useful in thepractice of the present invention include esters, alcohols, aliphaticnitriles, acyclic and cyclic aliphatic ethers, aliphatic ketones, anddipolar aprotic solvents.

[0016] Esters useful in the practice of the present invention have thegeneral formula R₁—C(O)O—R₂, wherein R₁ is H or linear or branched C1-6alkyl, and R₂ is linear or branched C1-6 alkyl. Examples of estersinclude methyl acetate, ethyl acetate, n-propyl acetate, iso-propylacetate, n-butyl acetate, iso-butyl acetate, methyl formate, n-propylformate, iso-propyl formate, n-butyl formate, and iso-butyl formate, tomention just a few. Alcohols (alkanols, glycols, and aromatic alcohols)useful in the practice of the present invention include methanol,ethanol, n-propanol, iso-propanol, ethylene glycol, propylene glycol,polyethylene glycol, polypropylene glycol, amyl alcohol and benzylalcohol, to mention just a few.

[0017] Aliphatic ketones useful in the practice of the present inventionhave the general formula R₁—C(O)—R₂, wherein R₁ and R₂ are,independently, linear or branched alkyl groups, each having from 1 to 4carbon atoms. Examples of aliphatic ketones include acetone, methylethyl ketone, and methyl iso-butyl ketone, to mention just three.

[0018] Examples of aliphatic nitriles useful in the practice of thepresent invention include acetonitrile, propionitrile, andbutyronitrile, to mention just three.

[0019] Ethers useful in the practice of the present invention includeboth acyclic and cyclic aliphatic ethers. Acyclic aliphatic ethers havethe general formula R₁—O—R₂, wherein R₁ and R₂ are as defined above.Examples of acyclic aliphatic ethers include diethyl ether, di-n-propylether, and ethyl n-propyl ether, to mention just a few. Tetrahydrofuranand the dioxanes are examples of cyclic aliphatic ethers useful in thepractice of the present invention.

[0020] Dipolar aprotic solvents are well known to the skilled artisan.Dimethyl acetamide (DMAC), dimethyl formamide (DMF),N-methyl-2-pyrrolidone (NMT), acatamide, dioxane and dioxalane areexamples of dipolar aprotic solvents useful in the practice of thepresent invention.

[0021] Hydrocarbon solvents are organic compounds, normally liquid atambient temperature, that are poor solvents for macrolides. Thehydrocarbon solvents can be aliphatic hydrocarbon solvents, or they canbe aromatic hydrocarbon solvents.

[0022] The aliphatic hydrocarbon solvents can be acyclic or they can becyclic. Acyclic hydrocarbon solvents can be linear or branched and havethe general formula C_(n)H₂₊₂, where n is from about 5 to about 10.n-Hexane, n-heptane, octane and iso-octane are examples of preferredacyclic aliphatic hydrocarbon solvents. Cyclohexane andmethylcyclohexane are examples of cyclic aliphatic hydrocarbon solvents.Examples of aromatic hydrocarbon solvents include benzene, toluene, thexylenes, and the tetralins, to mention just a few.

[0023] Any base, organic or inorganic, can be used in the practice ofthe present invention. Examples of inorganic bases include ammonia,alkali and alkaline earth metal hydroxides, bicarbonates, andcarbonates, to mention just a few. The amines are examples of organicbases that can be used in the practice of the present invention.

[0024] The present invention provides a method for crystallization andpurification of a macrolide, preferably tacrolimus, sirolimus,pimecrolimus, or everolimus including the steps of: providing, in acrystallization vessel, a combination of a macrolide starting material,a polar solvent, a hydrocarbon solvent, and water, whereby a water richphase is formed. A water-rich phase is a phase in with the majority ofthe solvent is water and can contain other solvents and solutes. The pHof the water-rich phase is or is adjusted to be about 7 or above,preferably about 8 or above. The pH can be adjusted by addition of base.

[0025] The combination is provided, preferably with agitation, and ismaintained at a temperature of between about −15° C. to about 50° C.,preferably between about −5° C. to about 40° C., most preferably betweenabout −2° C. to about 35° C. for at least about 1 hour, preferablybetween about 48 to about 100 hours, whereby a macrolide-rich phaseforms.

[0026] The manner in which the provided combination is assembled isirrelevant to the practice of the present invention. The components ofthe combination can be assembled in any order, or they can be assembledsimultaneously.

[0027] The combination of macrolide, polar solvent, hydrocarbon solvent,and water is provided in a crystallization vessel (crystallizationspace) provided with an agitator. The design and peculiarcharacteristics of the crystallization vessel are unimportant and theskilled artisan will know to select the crystallization vessel andagitator based on, among other things, the volume of the combination andthe process variables.

[0028] At the start of the first crystallization time, the combinationprovided will include two or more phases, at least one of which iswater-rich. The pH of the water-rich phase is about 7 or above,preferably about 8 or above. The pH of the water-rich phase can beconstant throughout the total crystallization time, or it can be variedin the course of the crystallization time, provided the pH is always atleast about 7 or above.

[0029] The desired pH is established with the use of any availableinorganic or organic base and the desired pH can be established in anymanner or sequence. For example, the pH of the water used to assemblethe combination can be adjusted, prior to assembly of the combination,with an inorganic or organic base. Thus, as used herein in connectionwith the combination provided, “water” will be understood to includedilute aqueous solutions (water solutions) of inorganic or organicbases, e.g., N/10 NaOH_(aq), N/10 KOH, N/10 Ca(OH)₂, N/10 NH_(3aq), N/10(C₂H₅)₃N_(aq), N/10 diethylamine or triethyl amine, N/10 pyridine etc.Base can be added before the water-rich phase is established by, forexample, admitting a low-boiling amine, e.g. methylamine, before wateris introduced. The skilled artisan will recognize a plethora ofalternatives to establishing the desired pH of the water-rich phase.

[0030] The pH can be adjusted after the combination is assembled byadding inorganic base, neat, especially as a gas, or in solution in asuitable solvent, e.g. water. The pH can be adjusted in increments. Forexample, the pH of the water used to assemble the combination can beadjusted to, e.g., ca. 7 before the combination is assembled and, afterassembly, the pH of the water-rich phase can be further adjusted, e.g.to pH 8, by the addition of base, neat or in solution.

[0031] During the course of the total crystallization time, at least onemacrolide-rich phase develops, from which the macrolide crystallizes,substantially free of impurities. At the end of the totalcrystallization time, crystalline macrolide is isolated by any of thecommon methods, for example filtration (gravity or pressure-assisted) orcentrifugation, to mention just two. The purity of the isolatedcrystalline macrolide rivals that of macrolide purified by multiple-passchromatography.

[0032] In one embodiment, the combination provided is assembled by thesteps of providing macrolide starting material that is a solution ofmacrolide, or a concentrate from macrolide extraction, preferablytacrolimus, sirolimus, pimecrolimus, or everolimus in a polar solventand combining the solution, in any order, with hydrocarbon solvent andwater.

[0033] The solution provided can be made by any means or method. Theconcentration of the solution provided is not critical and willgenerally be between about 0.05 g/mL (g macrolide per mL polar solvent)and about 0.3 g/mL. The macrolide can come from any source and can be asolid, semi-solid, or an oil (especially an oil that is a residue fromconcentration of extract from a whole-broth extraction ofmacrolide-containing biomatter).

[0034] The relative volumes of solution, water, and hydrocarbon solventare not critical. Typically, the ratio of the volume of solution to thevolume of hydrocarbon solvent will be between about 1:2 and about 1:10.The ratio of the volume of solution to the volume of water willtypically be between about 1:8 to about 1:25.

[0035] The pH of the water-rich phase can be adjusted and thecombination treated as described above.

[0036] In another embodiment, the combination provided is assembled bycombining, in any order, macrolide starting material, preferablytacrolimus, sirolimus, pimecrolimus, or everolimus starting material,hydrocarbon solvent, polar solvent, and water, wherein the tacrolimusstarting material is an oily phase that is a concentrate obtained byconcentrating a solution obtained by extracting macrolide-containingbiomatter with a hydrophobic extraction solvent, especially wherein thehydrophobic extraction solvent is selected from the group consisting ofC2-C6 linear and branched esters of acetic acid or formic acid, C3-C6linear or branched aliphatic ketones, halogenated methanes, and aromatichydrocarbons that are liquid at 25° C. and that have a boiling point atatmospheric pressure less than about 150° C., wherein the extraction isat a temperature between about 2° C. to about 70° C., especially betweenabout 30° C. and about 70° C., and at a pH of between about 5.5 andabout 13, especially between about 7.5 and about 13, to obtain thesolution of the macrolide in the hydrophobic extraction solvent.

[0037] The oil (macrolide starting material) can first be combined withpolar solvent or hydrocarbon solvent or water. The order is irrelevantto the practice of the present invention. The base required to establishthe desired pH can be introduced at any point, or at several pointsprior to or during the crystallization time. The base can be introducedneat, or as a solution, e.g. a solution in water.

[0038] The present invention, in certain of its embodiments, isillustrated by the following non-limiting examples.

EXAMPLE 1

[0039] Extraction:

[0040] Fermentation broth (22.2 m³) containing tacrolimus (3.42 kg) wasextracted with 6.4 m³ iso-butyl acetate at pH between 9.0-9.5. Theiso-butyl acetate solution was washed with water at pH between 6.0-8.0.The washed iso-butyl acetate phase was concentrated to oily-like residueunder reduced pressure at temperature between 40-45° C.

[0041] The oily-like residue was dissolved with iso-butyl acetate to avolume of 31 L. This concentrate was diluted with 167.5 L methanol and18.6 L water. The water-methanol solution was washed with 139.6 Ln-Hexane. The water-methanol phase was concentrated under reducedpressure to volume of 44 L, and the concentrate was diluted with 44 Lwater.

[0042] The obtained mixture was extracted with 88 L ethyl acetate. Theethyl acetate extract was concentrated to volume of 22.4 L.

[0043] Crystallization:

[0044] This concentrate of ethyl acetate extract was combined with 158.4L 0.1 M aqueous triethyl amine solution and with 67.3 L n-Hexane. Themixture was stirred at 20°-25° C. for 3 hours. The mixture was let tostand at 0°-25° C. for 48 hours (1 minute stirring every hour).

[0045] The crystals formed were isolated by filtration and weresuspended first in 83 L 0,1 M aqueous triethyl amine solution and,second, in 83 L n-Hexane. The crystals were isolated by filtration.

[0046] The crystals were dried at 40° C. under reduced pressure. Thedried crude tacrolimus had an assay 83%. Crude product contains 1.9 kgtacrolimus.

[0047] The yield of the crystallization step was 91%.

EXAMPLE 2

[0048] In the following example, a macrolide (tacrolimus), as an oilyconcentrate from whole-broth extraction of macrolide-containingbiomatter, was combined with polar solvent, hydrocarbon solvent, andwater containing a base. The combination was held at a crystallizationtemperature for a total crystallization time. At the end of the totalcrystallization time, the crystalline macrolide was isolated. Theproportions of components, the process variables, and the results arecollected in Table I. TABLE I Number of Tacrolimus Polar HydrocarbonTotal experiment Concentrate content solvent solvent Water t_(C)(hr)T_(C)(° C.) Yield Assay 1 15.23 g  1.42 g Ethyl acetate n-Hexane 0.1 NNaOH 24 +50-+20 41.82% 84.65% 30.3 ml  60.7 ml   273 ml 2 14.36 g  1.42g Ethyl acetate n-Hexane 0.1 N NaOH 20 +25-0 78.48% 81.68% 12.3 ml  73.7ml   172 ml 3 13.67 g  1.42 g Ethyl acetate n-Hexane 0.1 N NaOH 20 +25-079.06% 81.74%  7.5 ml  74.5 ml   164 ml 4 12.35 g  1.42 g Ethyl acetaten-Hexane 0.1 N NaOH 11 +20-−10 82.8% 82.85% 10.6 ml  63.4 ml   148 ml 511.47 g  1.42 g Ethyl acetate n-Hexane 0.1 N NH₃ 62 +25-0 79.19% 82.55% 9.8 ml   59 ml  137.6 ml 6 11.72 g  1.42 g Ethyl acetate n-Hexane 0.1 NNH₃ 62 +25-0 82.84% 79.20%  6.4 ml  63.9 ml 140.64 ml 7 12.17 g  1.42 gEthyl acetate n-Hexane 0.1 N (C₂H₅)₃N 62 +25-0 85.76% 85.03% 10.4 ml 62.6 ml  144.2 ml 8 12.94 g  1.42 g Ethyl acetate Cyclohexane 0.1 N(C₂H₅)₃N 62 +25-0 82.98% 82.18%   7 ml  70.6 ml  155.3 ml 9 13.28 g 1.42 g Ethyl acetate n-Hexane 0.1 N (C₂H₅)₃N 50 +25-0 72.84% 75.64%11.4 ml  68.3 ml  159.4 ml 10 14.72 g  1.42 g Ethyl acetate n-Hexane 0.1N (C₂H₅)₃N 32 +25-0 74.74% 84.81% 12.6 ml  75.8 ml   176 ml 11 11.36 g 1.42 g Ethyl acetate n-Hexane 0.1 N (C₂H₅)₃N 50 +25-0 71.64% 80.89% 9.7 ml  58.4 ml  136.3 ml 12 11.39 g  1.42 g Ethyl acetate n-Hexane 0.1N (C₂H₅)₃N 50 +25-0 88.32% 83.68%  9.8 ml  58.6 ml  136.7 ml 13 20.93 g 2.23 g Ethyl acetate n-Hexane 0.1 N (C₂H₅)₃N 62 +25-0  91.2% 86.49%17.9 ml 107.8 ml  251.2 ml 14 20.17 g  2.23 g Ethyl acetate n-Hexane 0.1N (C₂H₅)₃N 62 +25-0  62.7% 83.34% 17.3 ml 103.7 ml   242 ml 15 19.15 g 2.23 g Ethyl acetate n-Hexane 0.1 N (C₂H₅)₃N 62 +25-0  91.2% 88.05%16.4 ml  98.5 ml  229.8 ml 16  20.4 g  2.23 g Ethyl acetate n-Hexane 0.1N (C₂H₅)₃N 62 +25-0  91.2% 88.06%  8.7 ml  52.5 ml  122.4 ml 17 18.78 g 2.23 g Ethyl acetate n-Hexane 0.1 N (C₂H₅)₃N 62 +25-0 86.64% 86.90%   4ml  24.2 ml    56 ml 18  4.56 g 0.557 g Acetonitrile n-Hexane 0.1 N(C₂H₅)₃N 18 +25-+20 79.92% 83.46%  3.9 ml 23.45 ml  54.7 ml 19  4.62 g0.557 g n-Butanol n-Hexane 0.1 N (C₂H₅)₃N 18 +25-+15 63.12% 88.72% 3.96ml 23.76 ml  55.44 ml 20  4.58 g 0.557 g Acetone n-Hexane 0.1 N (C₂H₅)₃N18 +30-+20 87.07% 82.56% 3.93 ml 23.55 ml  54.96 ml 21  4.62 g 0.557 gIsobutanol n-Hexane 0.1 N (C₂H₅)₃N 18 +25-+10 67.34% 89.78% 3.75 ml 22.5 ml  52.44 ml 22  4.84 g 0.557 g Isopropanol n-Hexane 0.1 N(C₂H₅)₃N 18 +25-+20 80.26%   83% 4.15 ml  24.9 ml  58.08 ml 23  4.54 g0.557 g Ethanol n-Hexane 0.1 N (C₂H₅)₃N 18 +35-+20 76.92% 82.13% 3.89 ml23.35 ml  54.48 ml 24  4.43 g 0.525 g n-Propanol n-Hexane 0.1 N (C₂H₅)₃N18 +25-+15 75.6% 84.79% 3.79 ml 22.78 ml  53.16 ml 25  4.34 g 0.525 gMethanol n-Hexane 0.1 N (C₂H₅)₃N 18 +25-+20 77.16% 78.18% 3.72 ml 22.32ml  52.08 ml 26  3.84 g 0.525 g Diisopropyl n-Hexane 0.1 N (C₂H₅)₃N 18+25-+10 59.52% 72.35% ether 19.74 ml  52.08 ml 3.29 ml

EXAMPLE 3

[0049] Fermentation broth containing ascomycin was processed accordingto example 1. The process resulted in 60% yield for crude ascomycin.

1. A method of crystallizing a macrolide from a macrolide startingmaterial comprising the steps of: a) combining a macrolide startingmaterial, a polar solvent, a hydrocarbon solvent, and water, whereby atleast two phases are formed, at least one of which is a water-richphase, and wherein the pH of the water-rich phase is at least about 7,b) maintaining the combination at for at least 1 hour, whereby amacrolide-rich phase is formed from which the macrolide crystallizes. 2.The method of claim 1 further comprising the step of isolating themacrolide that crystallizes.
 3. The method of claim 1 wherein thecombination of step b is maintained at a temperature of from about −15°C. to about 50° C.
 4. The method of claim 3 wherein the combination ofstep b is maintained at a temperature of from about −5° C. to about 40°C.
 5. The method of claim 4 wherein the combination of step b ismaintained at a temperature of from about −2° C. and about 35° C.
 6. Themethod of claim 1 wherein the combination of step b is maintained forbetween 48 and 100 hours.
 7. The method of claim 1 wherein the polarsolvent is selected from the group consisting of alcohols, esters,nitrites and ethers.
 8. The method of claim 7 wherein the polar solventis selected from the group consisting of ethyl acetate, acetonitrile,methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol,acetone, diisopropyl ether, dimethyl formamide, and dimethyl acetamide.9. The method of claim 8 wherein the polar solvent is ethyl acetate. 10.The method of claim 1 wherein the hydrocarbon solvent is selected fromthe group consisting of n-hexane, n-heptane, octane, iso-octanecyclohexane, methylcyclohexane, benzene, toluene, and xylene.
 11. Themethod of claim 10 wherein the hydrocarbon solvent is n-hexane.
 12. Themethod of claim 1 wherein the pH of the water-rich phase is about 8 orhigher.
 13. The method of claim 1 wherein the water comprises a baseselected from NaOH, KOH, Ca(OH)₂, NH₃, Et₃N, diethylamine and pyridine.14. The method of claim 1 wherein the macrolide is selected from thegroup consisting of tacrolimus, sirolimus, pimecrolimus, everolimus, andascomycin.
 15. A method of crystallizing a macrolide from a macrolidestarting material comprising the steps of: a) combining a concentrateresidue from whole-broth extraction of macrolide-containing biomatter ina polar solvent with a hydrocarbon solvent, and water, whereby at leasttwo phases are formed, at least one of which is a water-rich phase, andwherein the pH of the water-rich phase is at least about 7, b)maintaining the combination at for at least 1 hour, whereby amacrolide-rich phase is formed from which the macrolide crystallizes.16. The method of claim 15 further comprising the step of isolating themacrolide that crystallizes.
 17. The method of claim 15 wherein thecombination of step b is maintained at a temperature of from about −15°C. to about 50° C.
 18. The method of claim 17 wherein the combination ofstep b is maintained at a temperature of from about −5° C. to about 40°C.
 19. The method of claim 18 wherein the combination of step b ismaintained at a temperature of from about −2° C. and about 35° C. 20.The method of claim 15 wherein the combination of step b is maintainedfor between 48 and 100 hours.
 21. The method of claim 15 wherein thepolar solvent is selected from the group consisting of alcohols, esters,nitrites and ethers.
 22. The method of claim 21 wherein the polarsolvent is selected from the group consisting of ethyl acetate,acetonitrile, methanol, ethanol, n-propanol, iso-propanol, n-butanol,iso-butanol, acetone, diisopropyl ether, dimethyl formamide, anddimethyl acetamide.
 23. The method of claim 22 wherein the polar solventis ethyl acetate.
 24. The method of claim 15 wherein the hydrocarbonsolvent is selected from the group consisting of n-hexane, n-heptane,octane, iso-octane cyclohexane, methylcyclohexane, benzene, toluene, andxylene.
 25. The method of claim 24 wherein the hydrocarbon solvent isn-hexane.
 26. The method of claim 15 wherein the pH of the water-richphase is about 8 or higher.
 27. The method of claim 15 wherein the watercomprises a base selected from NaOH, KOH, Ca(OH)₂, NH₃, Et₃N,diethylamine and pyridine.
 28. The method of claim 15 wherein themacrolide is selected from the group consisting of tacrolimus,sirolimus, pimecrolimus, everolimus, and ascomycin.
 29. A method ofcrystallizing a macrolide from a macrolide starting material comprisingthe steps of: a) combining, at a temperature of about 20° to about 25°C., macrolide starting material, ethyl acetate, n-hexane, and a watersolution of a base selected from NaOH, KOH, Ca(OH)₂, NH₃, (C₂H₅)₃N,diethylamine and pyridine whereby at least two phases are formed, one ofwhich is a water-rich phase, wherein the pH of the water-rich phase is >about 7, b) maintaining the combination at a temperature of about 20° C.to about 25° C. for at least 1 hour, whereby a macrolide-rich phase isformed from which macrolide crystallizes, c) maintaining the combinationat a temperature of about 0° C. to about 20° C. for at least 1 hour, andd) recovering the macrolide that crystallizes.
 30. The method of claim29 wherein the macrolide is selected from the group consisting oftacrolimus, sirolimus, pimecrolimus, everolimus, and ascomycin.
 31. Themethod of claim 29 wherein the pH of the water-rich phase is about 8 orhigher.
 32. A method of crystallizing a macrolide from a macrolidestarting material comprising the steps of: a) combining, at atemperature of about 20° to about 25° C., a concentrate residue fromwhole-broth extraction of macrolide-containing biomatter in ethylacetate, n-hexane, and a water solution of a base selected from NaOH,KOH, Ca(OH)₂, NH₃, (C₂H₅)₃N, diethylamine and pyridine whereby at leasttwo phases are formed, one of which is a water-rich phase, wherein thepH of the water-rich phase is > about 7, b) maintaining the combinationat a temperature of about 20° C. to about 25° C. for at least 1 hour,whereby a macrolide-rich phase is formed from which macrolidecrystallizes, c) maintaining the combination at a temperature of about0° C. to about 20° C. for at least 1 hour, and d) recovering themacrolide that crystallizes.
 33. The method of claim 32 wherein themacrolide is selected from the group consisting of tacrolimus,sirolimus, pimecrolimus, everolimus, and ascomycin.
 34. The method ofclaim 32 wherein the pH of the water-rich phase is about 8 or higher.35. In a method for crystallizing a macrolide from a macrolide startingmaterial, the step of combining the macrolide starting material, a polarsolvent, a hydrocarbon solvent, and water, whereby at least two phasesare formed, at least one of which is water rich, wherein the pH of thewater-rich phase is at least about
 7. 36. In a method for crystallizinga macrolide from a concentrate residue from whole-broth extraction ofmacrolide-containing biomatter in a polar solvent, the step of combiningthe macrolide concentrate in the polar solvent, a hydrocarbon solvent,and water, whereby at least two phases are formed, at least one of whichis water rich, wherein the pH of the water-rich phase is at least about7.